In recent years, human genome DNA mapping has been substantially completed, and research aimed at elucidation of gene functions has been actively conducted. It is necessary to specifically and extensively detect genes and proteins in vivo, and development of techniques for gene and protein detection has made progress worldwide. Meanwhile, a technique of identifying pathogens or viruses that have entered into organisms at the gene or protein level has heretofore been examined, and practical application of such technique has become possible. A variety of biosensors have been used as means for detecting biomolecules such as given genes or proteins in accordance with the intended purpose. The most common type of biosensor comprises a probe molecule that reduces the size of a biomolecule fixed to a solid surface. When a nucleic acid is to be captured, a nucleic acid is mainly used as a probe molecule. When a protein is to be captured, a protein is mainly used as a probe molecule. A biosensor comprising probe molecules fixed to a substrate is advantageous in that various types of probe molecules can be fixed to the same substrate via spotting system, ink-jet system, or other means. With the use of a biosensor substrate of such type, various biomolecules can be simultaneously subjected to extensive analysis, and such analysis can be completed rapidly. Representative examples of biosensors utilizing substrate surfaces include biomolecule-detecting elements, such as DNA microarrays or protein chips.
In recent years, methods of gene sequencing, such as with single-molecule-based sequencing utilizing arrays of single polynucleotide molecules (e.g., sequencing by synthesis (SBS)), have been disclosed as described in Patent Document 1 or Non-Patent Document 1, aimed at significant improvement in accuracy of gene expression analysis, as in the case of DNA microarray analysis. According to such technique, analyte polynucleotides modified with adequate primers are fixed to the substrate surface, and the resultant is used as a template to execute extension of each nucleotide with the use of a polymerase to construct complementary strands of the analyte polynucleotides. In each step of single-nucleotide extension, a fluorescent dye is introduced into the purine skeleton, the pyrimidine skeleton, or the end of a 3-phosphoric acid group, respectively, of each of the 4 different types of nucleotides. By conducting fluorescent detection in every step of extension for each single molecule, the nucleotide that has been introduced is distinguished. This step is repeated to decode the sequence of each single polynucleotide fixed site, and extensive analyte sequence information is obtained. In such a case, it is important to detect molecules with a high S/N ratio and improve the accuracy of sequencing. Since fluorescent information emitted from an enormous number of single polynucleotide fixed sites is detected with a CCD camara according to this technique, the average density for fixing polynucleotide molecules is determined in accordance with the pixel size of the CCD camera. Specifically, the average density for polynucleotide fixation or pixel resolution is regulated so as to capture a fluorescent signal from a single polynucleotide with a single pixel to as great an extent as possible. The pixel size is sub-micron (square) or greater when the spatial resolution of the optical detection system is taken into consideration.
In order to detect small quantities of DNA samples via sequencing as described above, it is necessary to improve the sensitivity of fluorescent detection. When fluorescent substances or luminescent substances that are not the targets of detection enter into the fluorescent detection region, fluorescence or luminescence emitted therefrom would be detected. When free fluorophores, impurities in a sample solution, or other substances adsorb to the planar surface of the evanescent field boundary in a non-specific manner, in particular, it would be difficult to distinguish fluorescence or luminescence emitted or light scattered from such non-specific absorptive materials from fluorescence or luminescence emitted from analytes. This may disadvantageously lower the fluorescent detection sensitivity or the accuracy of analysis. While such non-speicific absorptive materials can be avoided to some extent by coating a substrate surface or by other means, it is impossible to completely avoid such non-speicific absorptive materials. Thus, a method involving the use of technique for potentiation of fluorescence aimed at improvement in fluorescent detection sensitivity is reported in Non-Patent Document 2. In this case, silver nanoparticles resulting from modification of DNA probe molecules are fixed to a substrate and allowed to react with molecules in fluorescence-labeled analytes. When an excitation light is applied in order to detect the reaction amount, free electrons of the silver nanopartices cause local plasmon resonance, and fluorescence is potentiated. Sensitivity can be improved via such phenomena.
Fluorescence-enhanced fields are provided on the substrate in a grid-like manner at equal intervals, a single polymerase molecule is fixed to the fluorescence-enhanced field, and DNA is then subjected to extension. Since fluorescence excited by an evanescence is potentiated by the fluorescence-enhanced field, the S/N ratio or S/B ratio for fluorescent detection becomes sufficiently high even if non-specific adsorption takes place. However, variations occur in fine metal particle sizes, which may cause differences in the fluorescence potentiating effects among individual fine metal particles, and detection accuracy remains problematic. While a probe molecule is fixed to a fine metal particle by the liquid phase method, the site of fixation is determined at random. Thus, a probe molecule may be fixed to a region between a fine metal particle and the substrate, and it may inhibit extension by polymerase.
When fluorescence-enhanced fields are provided in a grid-like manner, a construct made of a noble metal is formed into the fluorescence-enhanced field via lithography. Thus, variations in fluorescence-enhanced fields may be reduced. In such a case, a probe molecule may be directly and selectively fixed to the fluorescence-enhanced field. Alternatively, a fluorescence-enhanced field and a metal sufrace having a composition different from that of the substrate may be provided as scaffolds for probe molecule fixation, and probe molecules may be selectively fixed. For example, Non-Patent Document 3 discloses a method of providing a metal oxide surface of TiO2 at a position at which an avidin biomolecule is to be provided and forming a membrane for preventing non-specific adsorption on the other SiO2 (quartz) surface. In such a case, introduction of a metal oxide surface of TiO2 as a probe molecule scaffold is considered to make possible selective introduction of biomolecules into the fluorescence-enhanced field.